The present invention relates to the field of tests and/or method for identifying asymptomatic carriers of X-linked immunodeficiencies. More particularly, it relates to methods for identifying asymptomatic carriers of agammaglobulinemia (XLA), severe combined immunodeficiency (XSCID). It also relates to immunoproliferative syndrome (XLP), Wiscott-Aldrich syndrome (WAS) and hyper-IgM syndrome.
While for some genetic diseases, heterozygous carriers can be identified by laboratory tests or specific findings on physical examination, there are many diseases in which carriers appear to be normal in all respects. Such is the case with most X-linked immunodeficiencies, including XLA, XSCID, XLP, WAS, and hyper-IgM syndrome. A carrier detection test would be extremely valuable for both family counseling and planning medical treatment, including bone marrow transplantation, of potentially affected off-spring. Furthermore, knowledge of carrier status in affected pedigrees would greatly facilitate gene mapping, and ultimately cloning of the genes responsible for these diseases. At the present time, no reliable carrier detection tests for these diseases are available.
In order to learn more about X-linked genetic defects of various types, a number of researchers have investigated such defects using existing genetic markers such as G6PD isoenzymes. These researchers have sought those rare individuals who are both heterozygous for G6PD, and for the defect under investigation. When the carrier of a recessive genetic defect appears normal, G6PD may be used to determine which X chromosome is active in the cells under investigation. The use of G6PD isoenzymes as a genetic marker is based upon the Lyon hypothesis which states that random inactivation of one or the other of the two X chromosomes in each female cell occurs during embryonic development. As a consequence, the Lyon hypothesis teaches that the human female is a mosaic, that is, only one X chromosome or the other is expressed in each cell.
Evidence exists that non-random X chromosomal representation occurs in particular cell-types in carriers of diseases such as Wiscott-Aldrich syndrome. Gealey et al, "Oleic Exclusion of Glucose-6-Phosphate Dehydrogenase in Platelets and T Lymphocytes from a Wiscott-Aldrich Syndrome Carrier", The Lancet, pp. 63-65 (Jan. 12, 1980). Gealey et al reports that non-random X inactivation occurs in WAS carriers in certain tissue lineages, particularly T lymphocytes. Gealey et al conclude that normal random X inactivation is followed by separate WAS-defect-dependent selection events occurring in post-thymus lymphocytes and platelets or platelet precursors. However, Gealey et al suggest that this hypothesis is not necessarily consistent with all of the available observations, at least as to expressed abnormalities in secondary platelet aggregation. See also Prchal et al, "Wiskott-Aldrich Syndrome: Cellular Impairments and Their Implications for Carrier Detection", Blood 56(6):1048-1054 (1980).
G6PD has also been used to determine whether skin related defects may be X-linked. G6PD has thus been employed to study heterozygous defects wherein selection may favor the mutant allele in vivo. See Migeon et al, "Adrenoluekodystrophy: Evidence for X Linkage, Inactivation, and Selection Favoring the Mutant Allele in Heterozygous Cells", Proc. Natl. Acad. Sci., 78:5066-5070 ((1981).
Nyhan et al., (1970) Proc. Natl. Acad. Sci USA 65: 214-218, have suggested either that subjects heterozygous for the Lesch-Nyhan syndrome (a deficiency of the X linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT)) undergo inactivation of the X chromosome that is not random or that random X chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme. Females heterozygous at the G6PD locus and the PRT locus were found to exhibit only one form of G6PD in circulating erythrocytes, thereby providing evidence supportive of this hypothesis.
Of course, genetic defects may or may not be related to non-random X chromosome inactivation. In "X Inactivation Patterns in Two Syndromes with Probable X-Linked Dominant, Male Lethal Inheritance", by Wieaker et al, Clinical Genetics, 28:238-242 (1985) (Sept., 1985 cover date; received University of Pennsylvania Library, Nov. 13, 1985) X inactivation patterns for two defects found only in females, incontinentia pigmenti (IP) and Aicardi syndrome, was hypothesized. IP is a rare skin condition; Aicardi syndrome is a symptomatically diagnosable condition. Preferential inactivation of the X chromosome carrying the IP gene with a proliferative advantage of the cell population was suggested, and partially supported by showing that the same X chromosome is preferentially active in fibroblasts grown from normal and hyperpigmented skin of a girl with IP, but not in a girl with Aicardi syndrome. Wieaker et al cultured fibroblasts grown from normal and hyperpigmented skin of the girl with IP and fused with HPRT-deficient mouse RAG cells according to a standard polyethylene glycol fusion protocol. Hybrids were selected and isolated and analyzed for an X-linked restriction fragment length polymorphism for which she was known to be heterozygous. The probe was thus employed to distinguish the two chromosomes of the patient and to determine the identity of the human X chromosomes retained in somatic cell hybrids. The results were said to demonstrate unambiguous evidence for preferential X chromosome activity in the IP patient, suggesting that there was somatic selection against cells that expressed the IP gene.
Somatic cell hybrids have also been used with Southern blotting and restriction-fragment-length polymorphisms (RFLPs) to investigate X-linked defects of Lesch-Nyhan syndrome patients. Nussbaum et al, "A Three-Allele Restriction-Fragment-Length Polymorphism at the Hypoxanthine Phosphoribosyltransferase Locus in Man", Proc. Nat. Acad. Sci. USA, 80:4035-4039 (July, 1983). This paper, which is co-authored by one of the inventors hereof, is hereby incorporated by reference. It is of particular pertinence for its disclosure of materials and methods relating to cell lines, somatic hybrid studies and its preparation of DNA and Southern blotting experiments.
For other purposes, it is sometimes desirable to distinguish active from inactive X chromosomes. Fogelstein et al, "In Use of Restriction Fragment Length Polymorphisms to Determine the Clonal Origin of Human Tumors", Science, 227:642-645 (Feb. 8, 1985), report the use of two restriction endonucleases to distinguish active from inactive copies of a gene. This strategy was used to demonstrate that three human cancers were each monoclonal.
Although X-linked agammaglobulinemia (XLA) was one of the first immunodeficiencies described, the genetic defect responsible for this disorder has not yet been identified. See Bruton, "Agammaglobulinemia", Pediatrics, 9:722-28 (1952). XLA is characterized by the onset of recurrent bacterial infections in the first few years of life. Concentrations of serum immunoglobulins are markedly decreased and the number of B cells present in the peripheral circulation of affected males is less than one percent of normal. However, pre-B cells, the non-circulating precursors of B cells, (Conley, "B Cells in Patients with X-Linked Agammaglobulinemia", J. of Immunol. 134:3070-3074 (1985)) can be detected in the bone marrow of affected individuals in approximately normal numbers (Pearl et al, "B Lymphocyte Precursors in Human Bone Marrow: An Analysis of Normal Individuals and Patients with Antibody-Deficient States", J. of Immunol., 120:1169-1175 (1978)).
Congenital severe combined immunodeficiency (SCID) has a male to female ratio of 4 to 1, implying that 60% of the cases are caused by a gene defect on the X chromosome. SCID is known to result in abnormalities in both T and B cell immunity, but the nature of the defect at the gene level is entirely obscure. From the point of view of genetic counseling, it is unfortunate that even though the X linked form is by far the most common form of SCID, all XSCID carrier mothers have a completely normal phenotype, and no carrier detection tests are currently available.
Although much is known about the clinical course of patients suffering from X-linked immunodeficiencies, a need still exists for a reliable screening test for asymptomatic XLA and XSCID carriers who are impossible to detect using normal immunological techniques.